The mitochondrial alcohol dehydrogenase (ADHIII) of the yeast Saccharomyces cerevisiae is synthesized in the cytoplasm and transported into the mitochondria. The nuclear gene encoding this isozyme, ADH3, has been cloned by taking advantage of its homology to the previously cloned ADH1 and ADH2 genes, which code for the cytoplasmic isozymes. The complete nucleotide sequence of the gene will be determined by chemical and enzymatic sequencing techniques. The limits of the functional gene will be determined by S1 nuclease analysis to locate the 5 feet and 3 feet ends of its mRNA. The function of mitochondrial ADH in respiration and fermentation will be studied by gene disruption. The normal gene will be inactivated by recombinant DNA techniques and yeast transformation and the phenotype of the mutant lacking a functional ADH3 gene will be studied. The transport of ADHIII into and its location within the mitochondria will be studied by characterizing newly-synthesized ADHIII polypeptides. The identification and characterization of a putative precursor will be done using immunological and protein sequencing techniques. These studies will utilize a yeast strain which lacks ADH1 and ADH2 genes so that neither mRNA nor polypeptides representing ADHI and ADHIII are present. Protein sequence analysis of the putative precursor and the mature protein will be performed in order to identify their amino termini. The function of different regions of ADHIII for mitochondrial transport, processing and targetting to the correct intramitochondrial site will be studied. Initial fusions will be made between ADH2 and ADH3 to study the necessity and sufficiency of the amino terminus for transport. Subsequent fusions will be made between ADH3 and lac Z if the amino terminus is not sufficient. Yeast mutants altered in mitochondrial transport of ADHIII will be isolated either by selection for resistance to allyl alcohol or by selection for resistance to antimycin. Mutants altered in the signal sequence of ADHIII and in transport proteins will be identified and characterized. A selection or screening system will also be developed to isolate conditional lethal mutants which might have pleiotropic transport defects.